Zoledronic acid generates a spatiotemporal effect to attenuate osteoarthritis by inhibiting potential Wnt5a-associated abnormal subchondral bone resorption

This study aimed to determine the effects of zoledronic acid (ZOL) on OA in rats and explored the molecular mechanism of osteoclast activation in early OA. A knee OA rat model was designed by surgically destabilizing the medial meniscus (DMM). Seventy-two male rats were randomly assigned to Sham+phosphate-buffered saline (PBS), DMM+PBS, and DMM+ZOL groups; rats were administered with 100 μg/Kg ZOL or PBS, twice weekly for 4 weeks. After 2, 4, 8, and 12 weeks of OA induction, the thickness of the hyaline and calcified cartilage layers was calculated using hematoxylin and eosin staining, degenerated cartilage stained with Safranin O-fast green staining was evaluated and scored, tartrate-resistant acid phosphatase (TRAP)-stained osteoclasts were counted, changes in subchondral bone using micro-computed tomography were analyzed, and PINP and CTX-I levels were detected using enzyme-linked immunosorbent assay. Using these results, 18 male rats were randomly assigned to three groups. Four weeks after surgery, Wnt5a, RANKL, CXCL12, and NFATc1 protein levels were measured in subchondral bone using western blotting, and mRNA levels of genes related to osteoclastogenesis in subchondral bone were measured using quantitative polymerase chain reaction. Bone marrow-derived macrophages were isolated as osteoclast precursors, and cell differentiation, migration, and adhesion were assessed by TRAP staining and Transwell assays, revealing that DMM induced knee OA in rats. Progressive cartilage loss was observed 12 weeks after OA induction. Subchondral bone remodeling was dominated by bone resorption during early OA (within 4 weeks), whereas bone formation was increased 8 weeks later. ZOL suppressed bone resorption by inhibiting Wnt5a signaling in early OA, improved the imbalance of subchondral bone remodeling, reduced cartilage degeneration, and delayed OA progression. Additionally, ZOL delayed OA progression and reduced cartilage degeneration via a spatiotemporal effect in DMM-induced OA. Osteoclast activity in early OA might be associated with Wnt5a signaling, indicating a possible novel strategy for OA treatment.


Animals and ethics statement
week-old (adult) male Sprague Dawley rats (Laboratory Animal Center of Ning Xia Medical University, Yinchuan, IACUC-NYLAC-2020-51) weighing 287 ± 12 g were grouphoused on a 12 h light/dark cycle with food and water ad libitum. The Animal Care and Experiment Committee of Ning Xia Medical University approved all experimental procedures associated with this study (protocol no. 2020-0001), which proceeded according to the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All surgery was performed under sodium pentobarbital anesthesia, all rats were euthanized via an overdose of intraperitoneal sodium pentobarbital, and every effort was made to minimize the number and suffering of rats included in this study.

Experimental design
A total of 72 of 90 rats were randomly divided into sham+PBS, destabilizing the medial meniscus (DMM)+PBS, and DMM+ZOL groups. The rats were sacrificed at 2, 4, 8, and 12 weeks after OA induction (n = 6 per time point), and the effects of Zoledronic acid (ZOL) on cartilage and subchondral bone during progressed OA were assessed, the OA bone resorption stage was identified according to the results of Osteoarthritis Research Society International (OARSI) scores, cartilage morphology, osteoclast number in subchondral bone. Based on these results, another 18 rats were randomly divided into three groups (n = 6 per group) to assess osteoclast formation, migration and adhesion of bone marrow-derived macrophages (BMMs), expression of Wnt5a, RANKL, CXCL12, nuclear factor of activated T-cells (NFATc1), tartrate-resistant acid phosphatase (TRAP), cathepsin K (Ctsk) and to explore the relative molecular mechanism during early OA. Fig 1 shows the experimental design.

OA induction
Rats were acclimated for 1 week and anesthetized with 1% pentobarbital sodium in phosphatebuffered saline (PBS, 60 mg/Kg). DMM rat models were created as described [17]. Briefly, the right knee joint capsule of each rat in DMM group was exposed according to a medial parapatellar approach; subsequently, the medial meniscotibial ligament was transected with microscissors to destabilize the medial meniscus, and the medial meniscus was reflected proximally toward the femur. Finally, the capsule and skin were sutured using 5-0 synthetic absorbable suture. By contrast, each rat in the sham+PBS group underwent to the same procedure without transections of medial meniscotibial ligament and destabilization of medial meniscus. All rats were resuscitated after surgical anesthesia by placing them on electric blankets.

ZOL subcutaneous administration
Changes of osteoclast activity during OA progression in each group were analyzed. Rats in the DMM+ZOL group were subcutaneously injected with ZOL (100 μg/Kg, Aclasta 1 , Novartis Pharma Stein AG, Switzerland) in PBS twice weekly for 4 weeks after surgery. The same volume of PBS was administered to the sham+PBS and DMM+PBS groups.

Micro-computer tomography (CT) analysis
Knee joints of six rats in each group were fixed in 4% paraformaldehyde (PFA) for 48 h. Three-dimensional (3D) μCT images were acquired using a SKYSCAN1076 (Bruker, Billerica, MA, USA) with an isotropic voxel resolution of 10 μm under the following parameters: pixel size, 10 μm; tube potential (peak), 40 kVp; tube current, 250 μA with 0.6˚rotations. A portion (0.5 mm below the growth plate and 1 mm in height, 3.5 mm ventrodorsal length) of the loadbearing region at the medial tibial plateau was identified as a region of interest (ROI). The trabecular bone morphometry includes bone volume fraction (BV/TV), trabecular thickness (Tb. Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), and connectivity density (Conn.D) were calculated.

Measurement of serum biomarkers of bone turnover
Six rats from each group were fasted but had free access to water for at least 6 h before collecting blood. Blood collected from tail vein at each time point was clotted at 20˚C for 30 min, and then centrifuged at 4,000 × g for 20 min. Serum was then aliquoted and stored at -80˚C. Concentrations of N-terminal propeptide of type I procollagen (PINP) and carboxy-terminal telopeptide of type I collagen (CTX-I) were assayed using enzyme-linked immunosorbent assay (ELISA) kits (Lianshuo Biological Technology Co., Ltd., Shanghai, China), as described by the manufacturer.

Histology
After acquiring μCT images, knee samples of six rats in each group were decalcified in 10% disodium ethylenediaminetetraacetate dihydrate (E8030; Solarbio Co., Ltd., Beijing, China) for 4 weeks, embedded in paraffin, sectioned at 5 μm thickness along the coronal plane and stained with hematoxylin and eosin (HE) using a kit (G1005; Servicebio, Wuhan, China) at 25˚C. The sections were placed in xylene twice for 20 min, anhydrous ethanol twice for 5 min each, and then 75% alcohol for 5 min. The sections were stained with hematoxylin for 3 min, rinsed in running water for 10 min, dehydrated in 85% and 95% alcohol for 5 min each, and then stained with eosin for 5 min. The thickness of the hyaline (HC) and calcified (CC) cartilage layers, and total articular cartilage (TAC) were calculated as described [18]. Before staining with Safranin O-fast green and TRAP (G1053 and G1050, respectively; Servicebio), slides were deparaffinized as described above, and then stained with Fast Green for 5 min, washed, dehydrated, and stained with Safranin O for 2 min. Histopathologically stained cartilage were graded using OARSI scores, as described [19].
Sections were incubated in working fluid from TRAP staining kit for 2 h, washed thrice with distilled water, then TRAP-stained slides were stained with hematoxylin for 3 min. Mature osteoclasts were counted, and ratios of Oc.S/BS that represented bone resorption activity in subchondral bone were calculated. Regions from three sections per rat were visualized using a DP71 microscope with DP controller software (Olympus Optical Co. Ltd., Tokyo, Japan).

Immunohistochemistry
Coronal sections of three rats in each group were incubated overnight at 4˚C with primary antibodies (all diluted 1:100) against Wnt5a (55184-1-AP), CXCL12 (17402-1-AP; both from Proteintech Group Inc., Rosemont, IL, USA), and NFATc1 (ab264530; Abcam, Cambridge, UK). Sections were washed thrice for 5 min, incubated with goat anti-rabbit immunoglobulin G (IgG) polymer-conjugated horseradish peroxidase (HRP) for 1 h at 37˚C, and then color was developed by adding 3,3-diaminobenzidine (DAB) (ZLI-9018; Beijing Zhongshan Jinqiao Biotechnology Co., Beijing, China) and counterstained with hematoxylin. Positively stained cells were counted in whole areas of tibial subchondral bone per specimen, and five sequential specimens per rat in each group were assessed. All measurements proceeded in a blinded fashion.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Samples of three rats from each group were processed as described above for western blotting. Total RNA was extracted from proximal tibias without articular cartilage using Multisource Total RNA Prep Kits (AP-MN-MS-RNA-250, Axygen Inc., Union City, CA, USA). First-strand cDNA was synthesized using TransScript 1 All-in-One First-Strand cDNA Synthesis Kit (AT341-01; TransGen Biotech, Beijing, China) and an S1000 Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA). Target genes were amplified by RT-qPCR using Perfect Start™ Green qPCR SuperMix (Cat No. AQ602-21; TransGen Biotech, Beijing, China) and an Applied Biosystems7500 Fast Real-Time PCR System (Thermo Fisher Inc., Waltham, MA, USA) under the following conditions: 45 cycles of 94˚C for 30 sec, 54˚C for 30 sec, and 72˚C for 34 sec. Table 1 lists the primer sequences. Relative mRNA expression levels were quantified using the 2 -ΔΔCq method. The loading control was β-actin.

Assays of migration and adhesion of osteoclast precursors
Osteoclast migration was evaluated using Transwell assay kits (3422; Corning Inc., Corning, NY, USA), as described [21]. Osteoclast precursors (200 μl containing 1 × 10 6 cells/mL) were loaded into the upper chamber of 24-well Transwells and left for 6 h to migrate through 8 μm polycarbonate filters to the bottom chambers containing 600 μl α-MEM containing 10% FBS and 30 ng/mL M-CSF. Osteoclast precursors in the lower chamber were stained with crystal violet for 10 min and then counted using a Zeiss light microscope (Axio Observer 7; Carl Zeiss AG., Oberkochen, Germany). Osteoclast precursors (1 × 10 5 cells/well) were seeded into 96-well plates and incubated for 30 min in α-MEM containing 10% FBS and 30 ng/mL M-CSF [22]. Cells were washed thrice with PBS, fixed with 4% PFA at 20˚C for 15 min, and stained with crystal violet for 10 min. Cells that adhered to the bottom of the plates were counted using a Zeiss light microscope.

Statistical analysis
Data were analyzed using one-way and two-way factorial design analysis of the variations followed by Student-Newman-Keuls and Bonferroni post hoc tests. Quantitative data are expressed as the means ± standard deviation (SD). For OARSI scores, data were analyzed using Kruskal-Wallis test followed by Dunn's test. GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA) and Stata/MP 15.0 (College Station, Texas 77845 USA) were used for statistical analysis, and values P < 0.05 were significantly different.

DMM-induced abnormal subchondral bone remodeling is inhibited by ZOL
Representative μCT images of knee joints demonstrated that DMM resulted in increased bone resorption from 2-4 weeks and increased bone formation from 8-12 weeks after surgery, indicating abnormal remodeling in subchondral bone. In contrast, ZOL attenuated bone resorption at an early stage (within 4 weeks postoperatively) and delayed bone formation at the advanced stage (8 weeks postoperatively). Osteosclerosis occurred at the tibial metaphysis, possibly due to osteoclast inhibition by ZOL in active growth plates (Fig 2A). Based on trabecular bone morphometric parameters, subchondral bone was significantly altered in the DMM+PBS group compared that in the sham+PBS group in terms of decreased BV/TV (Fig 2B), Tb.N ( Fig 2E) and Conn.D (Fig 2F), reduced Tb.Th (Fig 2C), and increased Tb.Sp ( Fig 2D) at an early stage, and increased BV/TV, thickened Tb.Th, narrowed Tb.Sp, and decreased Tb.N and Conn.D at advanced stages. However, these changes were preserved by ZOL, especially during early OA. PINP and CTX-I levels consistently and time-dependently increased in the DMM +PBS group compared with those in the sham+PBS group; ZOL also decreased CTX-I levels in early OA (Fig 2G and 2H).

ZOL inhibits articular cartilage degeneration in DMM-induced OA rats
Sections stained with HE and Safranin O-fast green to assess histomorphological changes in cartilage during OA progression. Cartilage degeneration generally occurred and time-dependently worsened from 2-12 weeks after surgery. HE staining showed decreased thickness of the HC, whereas the CC increased with tidemark duplication and moved closer to the articular surface in the DMM+PBS group compared with that in the sham+PBS group (Fig 3A and 3B). The thickness of the CC was attenuated by ZOL at 2 and 4 weeks after surgery (Fig 3C). The CC/TAC ratio was higher in the DMM+PBS group than those in sham+PBS and DMM+ZOL PLOS ONE groups 4 weeks postoperatively; the sham+PBS group and DMM+ZOL groups did not significantly differ at 2 and 4 weeks after surgery (Fig 3D).
Safranin O-fast green staining indicated that compared with those in the sham+PBS group at 2 weeks following surgery, matrix and chondrocytes in the DMM+PBS group were lost mainly in the superficial cartilage layer. This extended into the deep zone and generated

PLOS ONE
irregular cracks at 4 weeks. Full-thickness degeneration and tidemark replication that occurred at 8 weeks, became more widespread by 12 weeks (Fig 4B). The cartilage degeneration grade was mild, and the process was slow in the DMM+ZOL group (Fig 4C). The OARSI score showed significantly increased cartilage degeneration in the DMM+PBS group, compared with that in the sham+PBS group, while degeneration was attenuated in the DMM+ZOL group after OA induction (Fig 4D).

ZOL attenuates osteoclast activity in early OA in vitro and in vivo
The number of TRAP-positive osteoclasts was higher in the DMM+PBS group than the sham +PBS group 4 weeks after OA induction (Fig 5A and 5B), whereas ZOL significantly inhibited osteoclast formation (Fig 5C and 5D). These findings supported the hypothesis that DMM caused subchondral bone loss by enhancing bone resorption mediated by increasing osteoclasts during early OA.
The histomorphological and μCT findings demonstrated accelerated bone turnover at the early stage of DMM-induced OA in rats, indicating that bone resorption mediated by osteoclasts increased. To confirm this, BMMs were isolated from tibial bone marrow 4 weeks after OA induction and osteoclast formation, migration, and adhesion were examined. The results of TRAP and crystal violet staining showed that DMM significantly enhanced, whereas ZOL inhibited osteoclast formation (Fig 6A and 6D), BMM migration (Fig 6B and 6E) and adhesion (Fig 6C and 6F).

ZOL attenuates expression of Wnt5a and osteoclastogenesis genes induced by DMM during early OA
To further elucidate the possible mechanism of osteoclastogenesis in early OA at the molecular level, the activities of Wnt5a and CXCL12, together with NFATc1, RANKL, Trap, Opg, and Ctsk in the tibia were examined. Immunohistochemical staining indicated that significantly more cells in the DMM+PBS group were positive for Wnt5a (Fig 7A and 7E), CXCL12 (Fig 7B  and 7E), and NFATc1 (Fig 7C and 7E), which play important roles in cell migration and osteoclastogenesis in tibial subchondral bone, than in the sham+PBS group. Western blotting ( Fig  7D and 7F) and qPCR (Fig 7G-7I) data of the proximal tibia showed that ZOL suppressed the significantly increased expression of Wnt5a, CXCL12, and osteoclastogenesis genes induced by DMM. These findings further supported the possible effects of Wnt5a on osteoclastogenesis and function.

Discussion
This study determined the spatiotemporal order of ZOL in OA and the underlying molecular mechanisms. These findings indicated that DMM induced OA with a higher OARSI score and CC/TAC ratio in model rats. Abnormal subchondral bone remodeling was characterized by enhanced bone resorption whereas osteoclast activity was significantly increased in early OA, which was confirmed by the CTX-I and PINP findings. Furthermore, ZOL alleviated bone resorption by inhibiting osteoclast activity, thus delaying OA progression (time) and reducing cartilage degeneration (space), reflecting the spatiotemporal effect of ZOL on OA. Additionally, osteoclast activation might be associated with Wnt5a signaling.
OA is a chronic, progressive, and degenerative disease characterized by abnormal subchondral bone remodeling and cartilage degeneration. The stage and severity of OA may be quantified using OARSI scores, which comprise the standard method for OA cartilage histopathology assessment [19]. Compared with other scoring methods, the OARSI score reveals early pathological changes in OA and provides guidance for basic research, as well as

PLOS ONE
OA clinical diagnosis and treatment [23]. Lost trabecular bone and cartilage degeneration may reportedly be determined in animal models using OARSI scores at 1 or 2 weeks after OA induction [24,25]. The results of the present study contradicted this, as only minor cartilage degeneration was observed 4 weeks after OA induction. This might be partially due to the fact that the OA model in the present study was induced using DMM, which is minimally invasive, and does not generate interference due to trauma, or acute inflammatory or immune responses, and thus, better simulates chronic, progressive, and degenerative OA. Sex and species may also affect OA [26,27].
The knee OA seems to involve the whole joint [28]. Articular cartilage and subchondral bone form structural and functional units that participate in OA pathology [29]. Traditionally, the subchondral bone plate has been regarded as a barrier to prevent molecular and informational communication between articular cartilage and subchondral bone [30]. However, recent findings have suggested that subchondral bone remodeling influences cartilage degeneration through a crosstalk mechanism in bone-cartilage junctions [31,32]. Experimental animal and clinical findings have proven that increased numbers of cysts in subchondral bone during early OA is an adaptive change in response to abnormal bone remodeling and is associated with osteoclast activation [33]. Consistent with previous results, more osteoclasts were found in subchondral bone and BMM migration and adhesion increased 4 weeks after DMM-induced OA, which enhanced bone resorption and reduced subchondral bone mass. However, others have focused on intermediate or late OA, and may have missed the crucial early stage in which subchondral bone undergoes rapid resorption, similar to the trigger for OA deterioration. The third-generation, nitrogen-containing, long-acting bisphosphonate, ZOL, significantly increases bone mineral density by suppressing osteoclast activity and it is the standard clinical treatment for osteoporosis [34]. Thus, ZOL was applied to rat models of OA. Early subchondral bone resorption was inhibited, and OARSI scores, as well as the TRAP staining, μCT, and ELISA results confirmed that ZOL improved the abnormal subchondral bone remodeling and then attenuated cartilage degeneration. We believe that this was a causal sequential event, as ZOL delayed OA progression and reduced cartilage degeneration, which was referred to as a spatiotemporal effect of ZOL on OA. These findings indicated that early effective measures to improve bone remodeling might be an OA treatment strategy.
The Wnt/β-catenin signaling pathway is involved in bone formation and cartilage degeneration in late OA [8]. It exerts bone remodeling behavior coupled with bone formation, regardless of whether bone resorption is associated with Wnt5a signaling pathways. Maeda et al. have found that mice deficient in Wnt5a have abnormal bone remodeling due to impaired osteoclast formation [12]. In contrast, another study has found that Wnt5a upregulates low-density lipoprotein receptor-related protein 5/6 expression in osteoblast-lineage cells, which promotes osteoblastogenesis and inhibits adipogenesis via Wnt/β-catenin signaling [12]. These previous findings suggest that Wnt5a does not simply inhibit, but rather enhances Wnt/β-catenin signaling; notably, Wnt5a exerts bidirectional regulation in tumors [11]. Therefore, it is inferred that Wnt5a derived from different cells in various microenvironments binds to a diversity of receptors to form different complexes, thus activating corresponding downstream transcription factors and playing a regulatory role. Here, DMM increased Wnt5a expression and upregulated levels of RANKL, CXCL12, NFATc1, and osteoclastogenesis genes, including TRAP and Ctsk, which were significantly attenuated by ZOL. Bone resorption of osteoclasts might have been inhibited by ZOL, which would have changed the acidic environment in subchondral bone and indirectly inhibited the expression of RANKL, CXCL12, and osteoclastogenesis mediated by the Wnt5a signaling pathway.
However, this study has several limitations. First, data were collected at four time points to investigate early subchondral bone loss and late-stage OA development. Inevitably, this excluded many time points during which key events related to OA might have occurred. Second, the same model was not tracked and data about technical limitations were not collected, such as generating μCT results from observations in vivo, which could also save experimental animals. Third, referring to previous study [35], we only selected a single dose of ZOL (100 μg/ Kg), whereas did not observe the effects of different dose and frequency of administration on OA. Fourth, 8-week-old rats were used for modeling in this study, although the observation lasted for 12 weeks after modeling, it was difficult to completely simulate the changes of OA in human. The primary OA is more likely to occur in the aged than in young people, and it may be that young people have a better capacity to manage joint damage. Moreover, in controlled experiments, the complexities of changing metabolism and skeletal size during growth can obscure how joints respond to damage and therapy. Thus, to obtain the most meaningful insights into human OA, it is essential to use skeletally mature animals whenever possible [36]. In contrast to primary degenerative OA, juvenile idiopathic arthritis (JIA) and rheumatoid arthritis (RA) are present from early childhood, and their pathogenesis may be related to infection, genetics and immunity [37]. Osteochondritis dissecans (OCD) is another adolescent disease characterized by focal, idiopathic changes in the subchondral bone and overlying articular cartilage. Repetitive microtrauma is the most common external mechanism that may lead to this disorder [38]. In addition, we only detected the relationship between Wnt5a and osteoclast differentiation related factors in subchondral bone tissue through animal experiments, and the specific molecular mechanism of their interaction needs to be further explored through cell experiments.

Conclusion
By inhibiting the bone resorption of osteoclasts in early OA, ZOL improves the imbalance of subchondral bone remodeling, thus delaying OA progression and reducing cartilage degeneration. This is regarded as a spatiotemporal effect of ZOL on OA, which may be consider as evidence of crosstalk between articular cartilage and subchondral bone. Osteoclast activity in early OA might be associated with the Wnt5a signaling pathway, which could serve as a strategy for the precise treatment of OA.
Supporting information S1 File. Details about some experiments. (PDF)